Porphyromonas gingivalis is a Gram-negative anaerobic bacterium (bacteroides) and a major causative agent of periodontal diseases. In addition, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to rheumatoid arthritis. Epidemiological studies demonstrate that this oral pathogen could have broad range of human health impact. Bacterial pathogens, including P. gingivalis, express and secret an array of virulence factors, which enable them to cause disease in human host. Particularly, P. gingivalis secretes strong proteases called gingipains that are implicated as virulence factors, i.e., the arsenal to fight against the host, in periodontal pathogenesis. A newly characterized type IX secretion system, PorSS, distinct from those characterized in other Gram-negative bacteria, has been linked to export of these gingipains. Two-component regulatory systems (TCS) provide the most ubiquitous signal transduction systems in bacteria. Pathogenic bacteria employ varied TCSs in response to host environments by interacting with signal molecules present in infected niches and regulating specific sets of genes including virulence determinants during infection eventually leading to disease. Our preliminary results have shown that P. gingivalis porX and porY loci encode a TCS histidine kinase (HK) and response regulator (RR), respectively. This unusual TCS is important for bacterial virulence because it activates transcription of at least 6 PorSS structural genes. This project aims to provide a detailed understanding of the regulatory roles of the PorX-PorY system in P. gingivalis gene expression including its regulatory mechanisms of action, contribution to bacterial virulence and the novel strategies employed to influence this signal transduction process. Since this TCS controls the expression of multiple key virulence factors of this clinically important oral pathogen, we will use high- throughput assays to genome-wide characterize the gene profile to specify the virulence determinants controlled by this regulatory system and reveal the physiological processes involved. We will carry out phosphoproteomic analysis to study how the phosphatase activity of PorX would dominant a phosphorelay mechanism to regulate specific target genes in this oral bacterium. Furthermore, we will address possible dual function of the CheY-like C terminal domain in PorX, which may be a singular example of a two-component system that can function as a transcriptional regulator, meanwhile, as an effector on a secretion machinery on the membrane. The PorX-regulated gene profile partially overlaps with those by the hemin-responsive HaeRS TCS. Thus, we will determine whether the PorX-PorY system can build up an integrated signaling circuit by cross-talking with the regulator HaeR in response to hemin. This study will be a new effort for understanding regulatory mechanisms of an unusual two-component system formed by orphan proteins PorX and PorY from P. gingivalis.